Hair dyeing premixes containing peroxidase enzymes stabilized with heme complexing agents

ABSTRACT

Premixes for dyeing hair comprising a peroxidase enzyme, an aromatic oxidation dye precursor, an anti-oxidant, and a heme complexing agent. The premix is combined with hydrogen peroxide on the hair whereupon a dye is formed and the hair is colored.

United States Patent [191 Kaiser [451 July 8,1975

[ HAIR DYEING PREMIXES CONTAINING PEROXIDASE ENZYMES STABILIZED WITH HEME COMPLEXING AGENTS Emil Thomas Kaiser, Chicago, Ill.

[52] US. Cl. 8/10.2; 8/10.1; 8/11;

8/32 [51] Int. Cl A6lk 1/12 [58] Field of Search 424/94; 8/10.2, 11, 32

[56] References Cited UNITED STATES PATENTS 2,539,202 l/195l Peck 8/10.2

3,200,040 8/1965 Lange 8/10.2 3,251,742 5/1966 Soloway 8/l0.2 3,488,138 1/1970 ls'cowitz 8/10.2 X

FOREIGN PATENTS OR APPLICATIONS R73,397 6/1960 France 424/94 Primary Examiner--Albert T. Meyers Assistant Examiner-Vera C. Clarke Attorney, Agent, or Firmt-Douglas C. Mohl; Ronald L. Hemingway; Richard C. Witte [5 7] ABSTRACT Premixes for dyeing hair comprising a peroxidase enzyme, an aromatic oxidation dye precursor, an antioxidant, and a heme complexing agent. The premix is combined with hydrogen peroxide on the hair whereupon a dye is formed and the hair is colored.

6 Claims, No Drawings HAIR DYEING PREMIXES CONTAINING PEROXIDASE ENZYMES STABILIZED WITH HEME COMPLEXING AGENTS BACKGROUND OF THE INVENTION The present invention relates to enzyme-activated hair dyeing premixes with improved stability.

Hair dyeing premixes containing a peroxidase enzyme, an aromatic oxidation dye precursor, and an anti oxidant such as sodium sulfite, are known in the art. See, for example, West German Pat. No. 2,155,390 which issued in 1971. However, solutions of such premixes have a shelf life of at most only a few days because the anti-oxidant which preserves the dye precursor also deactivates the enzyme. As hair dyeing premixes must have a shelf life longer than a few days in order to be commercially feasible, there is a need in the prior art for some means of stabilizing the enzyme against the anti-oxidant. The present invention satisfies this need.

It is known that peroxidase has one ferriprotoporphyrin group per molecule and that certain substances will combine with B ferriprotoporphyrin to form complex compounds. It is also known that peroxidase reacts with hydrogen peroxide to give a series of complexes in consecutive reactions in each of which a substrate is oxidized to a free radical. Further, an article by I. Fridovich entitled The Stimulation of Horseradish Peroxidase by Nitrogenous Ligands in the Journal of Biological Chemistry, Vol. 238, No. 12, 1963, pp. 3921-3927, discusses nitrogenous ligand enhancement of the peroxidation of dianisidine by peroxidase and hypothesizes that the nitrogenous ligands enhance peroxidase activity by combining rapidly and reversibly with one side of the hematin iron of the enzyme.

SUMMARY OF THE INVENTION The present invention lies in the discovery that peroxidase enzymes in combination with certain nitrogenous ligands are stabilized against some anti-oxidizing agents without loss of enzymatic activity. The present invention further lies in the discovery that a peroxidase enzyme-activated hair dyeing premix solution comprising peroxidase enzyme, at least one oxidation dye precursor and an anti-oxidant can be stabilized by certain nitrogenous ligands so that when combined with hydrogen peroxide on the hair after months of storage, the premix will be effective to form a dye and color the hair. Without the stabilizing effect of the nitrogenous ligand, the enzyme of the premix is deactivated in a few days by the reducing action of the anti-oxidant, resulting in an ineffective premix solution.

DESCRIPTION OF THE INVENTION The peroxidase enzyme-activated hair dyeing premix of the present invention comprises:

A. from about 0.005 ppm to about 500 ppm ofa peroxidase enzyme; B. from about 0.01 to about 1% of a nitrogenous 1igand; C. from about 0.001 to about 6% by weight of at least one aromatic compound which is an oxidation dye precursor;

D. from about 0.1 to about 1% of an anti-oxidant compound;

E. balance, water and minors, said composition having a pH of from about 4 to about 10.

Care must be taken so that the enzyme is not put into solution with the anti-oxidant unless the nitrogenous ligand is also present in the solution. The enzyme of the premix is stabilized against the reducing action of the anti-oxidant by the heme complexing agent, resulting in longer shelf life for the premix solution. It is intended idazole and 0.5% sodium dithionite. However, horseradish peroxidase retains only 0.7% of its original activity after 3 days in the presence of 0.5% sodium dithionite alone. Similarly, in Table II, horseradish peroxidase is stabilized against sodium sulfite by 0.1% benzimidazole.

TABLE I Percent Activity Remaining Time (Days) 0.484 mg/ml Horseradish Peroxidase 1 2 3 7 16 23 32 with 0.5% Na Dithionite 1.7 1.3 0.7 with 0.5% Na Dithionite 102 1 17 123 and 0.1% Benzimidazole 41 with 0.5% Na Dithionite 101 104 95 98 103 and 0.1% Imidazole 105 TABLE 11 Percent Activity Remaining Time (Days) 0.05 mg/ml Horseradish Peroxidase 3 7 with 0.5% Na same no data 58 with 0.5% Na Sulfite and 0.1% Benzir nid azole 93 100 The percentages in Tables 1 and II were determined from the absorbance change at 400 nm and 25C. of 100 .l, 0.1 M pyrogallol in 3 ml of .37: hydrogen peroxide in pH 6, 0.1 M phosphate buffer caused b y the addition of enzyme sample. The enzyme samples consisted of horseradish peroxidase enzyme with an R2 value of 114 in Table I and 1.1 in Table II, a pH 6, 0.1 M phosphate buffer and thn.- nitrogenous ligand and/or anti-ox1dant as shown in Tables I and II. Samples were stored at 80F. and yellow light was employed to prevent any photoinactivation.

Peroxidase enzymes suitable for use in the present invention are those which catalyze the oxidation of various materials (including the oxidation dye precursors herein) by hydrogen peroxide.

Peroxidase enzymes can be derived by known methods from a variety of plants, e.g., apples, apricots, barley, horseradish roots, beets, cabbage, carrots, corn, cotton garlic, grapefruit, mint. rhubarb, soybeans and spinach, and peroxidase enzymes can also be isolated from animal sources, e.g., bovine milk, or from bacterial sources, e.g., Acetobacter peroxidans.

Specific examples of these enzymes are NAD peroxidase which is derived, for example, from Staphylococcus faecalis, NADP peroxidase, which is derived, for example, from casei, fatty acid peroxidase, which is derived, for example, from peanuts, cytochrome peroxidase, which is derived, for example, from bakers yeast, peroxidase, which is derived, for example, from the horseradish root, iodinase, which is derived, for example, from thyroid gland tissue and glutathione peroxidase, which is derived, for example, from liver and blood. These enzymes are all well known and are classified in Class 1.11 [i.e., Class 1, Subclass ll of the Recommendations (1964) of the International Union of Biochemistry on the Nomenclature and Classification of Enzymes]. This system and a list of known enzymes classified according to it are set forth in Comprehensive Biochemistry, Florkin and Stotz, Vol. 13, Enzyme Nomenclature (1965), published by Elsevier Publishing Company, New York, NY. According to this system Class 1 indicates an oxidoreductase, and Subclass 11 indicates that the acceptor or substrate which is acted upon is hydrogen peroxide. Following the subclass number is a subsubclass number, which in turn is followed by the serial number of the enzyme in the sub-subclass. The enzymes recited above have the following classification numbers according to this system. NAD peroxidase (1.11.1.1), NADP peroxidase (1.11.1.2), fatty acid peroxidase (1.11.1.3), cytochrome peroxidase (1.11.1.5), peroxidase (1.11.1.7), iodinase (1.11.1.8) and glutathione peroxidase (1.11.1.9).

Although any of the aforementioned enzymes are suitable for use herein, the preferred peroxidase enzyme is the one called horseradish peroxidase. It has the classification number 1.11.1.7 and is sometimes simply called peroxidase. It is the most readily available of all the peroxidases.

The peroxidase enzymes herein can be used in their pure crystalline form, which is obtained by isolating the enzymes from other materials present during their preparation, or they can be used in a diluted form where the enzyme is present in a composition along these materials and/or added inert diluents.

Commercially available enzyme preparations normally contain the enzyme in combination with inert diluent and carrier materials such as carbohydrates, agglutinating proteins, inorganic salts such as sodium sulfate, calcium sulfate, and the like. In such preparations the enzyme constitutes a minor component and comprises from about 1 to about 50% by weight of the preparation. The remaining 50 to 99% is comprised of the hereinbefore described diluents and carriers. The commercially available enzyme-containing preparations are preferred as sources of enzyme herein as they are more readily available than the pure crystalline enzyme and provide known, pre-determined and desirable levels of enzyme activity.

In the coloring process herein, the peroxidase enzyme is used at concentrations of from about 0.005

ppm to about 500 ppm, and preferably from about 0.05 ppm to about ppm in the coloring solution. These levels are based on weight of pure enzyme. If a commercial enzyme preparation is used wherein the enzyme is combined with diluents and carriers, as hereinbefore described, the concentration of the enzyme preparation will have to be proportionately higher in order to achieve the required concentration of pure enzyme. The amount of pure enzyme present in such compositions can be readily determined by known assay methods.

The oxidation dye precursors which are used in the compositions and processes herein include aromatic diamines, various substituted phenols, amino phenols and derivatives of these aromatic compounds (e.g., N- substituted derivatives of the amines and ethers of the phenols). The oxidation dye precursors useful herein can be classified as primary oxidation dye precursors and secondary oxidation dye precursors, as detailed hereinafter. In general terms, oxidation hair dye precursors include those monomeric aromatic compounds which, on oxidation, form oligomers or polymers having extended conjugated systems of electrons in their molecular structure. Because ofthe new electronic structure, the resultant oligomers and polymers exhibit a shift in their electronic spectra to the visible range and appear colored. For example, oxidation dye precursors capable of forming colored polymers include materials such as various aromatic amines having a single functional group and which, on oxidation, form a series of conjugated imines and quinoid dimers, trimers, etc. ranging in color from green to black. Compounds, such as p-phenylenediamine, which have two functional groups are capable of oxidative polymerization to yield higher molecular weight colored materials having extended conjugated electron systems, i.e., the so-called Bandrowskis Base type of dye compound. Color modifiers, such as those detailed hereinafter as secondary oxidation dye precursors, can optionally be used in conjunction with the primary oxidation dye precursors herein and are thought to interpose themselves in the colored polymers during their formation and to cause shifts in the electronic spectra thereof, thereby resulting in changes in color and/or color intensity. It is to be understood that the peroxidase enzymes disclosed herein are suitable for use (in conjunction with a peroxide source, e.g., H 0 as detailed herein) with all manner of primary and secondary oxidation dye precursors. A representative list of oxidation dye precursors suitable for use herein is found in Sagarin, Cosmetic Science and Technology, lnterscience, pages 504 and 508, and the dye precursors detailed below are only by way of example and are not intended to limit the compositions and processes herein. Additional oxidation dye precursors useful herein are described in French application Pat. No. 1,318,072 and Fr. Addition 90,633, Jan. 19, 1968, to Schwarzkopf; British Pat. No. 1,127,080, Sept. 1 l, 1968, to Kalopissis and Bugaut; and Netherlands Application No. 6,609,833, Feb. 6, 1967, to Therachemie Chemisch Therapeutische G.m.b.l-l., incorporated herein by reference. Pyridine, quinoline, isoquinoline oxidation dye precursors such as those disclosed by Bergwein, Reichst., Aromen, Koerperpflegem. 17 (14) 136-8 (1967), are also suitable herein.

The oxidation dye precursors.which are used in the process of the present invention can be divided into two classes, primary and secondary. The primary oxidation dye precursors are essential to the practice of the invention and include those aromatic diamines, polyhydric phenols. aminophenols and derivatives of these aromatic compounds (e.g., N-substituted derivatives of the amines. and ethers of the phenols) which produce color formation in the following test, which is performed at room temperature (about 18 to 28C.).

PRIMARY DYE PRECURSOR TEST ml. of aqueous buffer (pH 5 to 8) containing 0.01 to 1.0% (by weight) H 0 is mixed with 0.1 to 1.0 ml. of a 1% (by weight) aqueous or alcoholic solution of the precursor. To this mixture is added an amount of horseradish peroxidase such that the final mixture contains 0.01 to 100 ppm peroxidase (based on weight of pure enzyme). The mixture is left standing to allow color formation. A suitable primary oxidation dye precursor will give color formation within 5 minutes. Some oxidation dye precursors, because of their self-color, impart a pale color to the solution before addition of enzyme. Color formation, in this test, refers to a visually perceptible color change which occurs after the addition of enzyme.

The aromatic diamines, polyhydric phenols, aminophenols, and derivatives thereof, described above as primary oxidation dye precursors, can also have additional substituents on the aromatic ring, e.g., halogen, aldehyde. carboxylic acid, nitro, sulfonic acid and substituted and unsubstituted hydrocarbon groups, as well as additional substituents on the amino nitrogen, and on the phenolic oxygen, e.g., substituted and unsubstituted alkyl and aryl groups.

Examples of aromatic diamines and derivatives thereof, aminophenols and derivatives thereof and polyhydric phenols and derivatives thereof, respectively, are compounds having the general formulas (A), (B) and (C) below:

wherein X is hydrogen, halogen, (etg. fluorine, chlorine, bromine or iodine), nitro, amino, hydroxyl,

COOM or SO M (where M is hydrogen or an alkali or alkaline earth metal, ammonium, or substituted ammonium wherein one or more hydrogens on the ammonium ion is replaced with a l to 3 carbon atom alkyl or hydroxyalkyl radical), wherein R R R and R are the same or different from each other and are selected from the group consisting of hydrogen, C to C alkyl or alkenyl and C to C aryl, alkaryl or aralkyl, and R is hydrogen, C to C alkyl or alkenyl (including substituted alkyl or alkenyl wherein the substituents are selected from those designated as X, above) or C to C aryl. alkaryl or aralkyl (including substituted alkyl, alkaryl or aralkyl groups wherein the substituents are selected from those defined as X, above). Specific examples of formula (A) compounds are:

o-phenylenediamine m-phenylenediamine p-phenylenediamine 2chloro-p-phenylenediamine 2-iodo-p-phenylenediamine 4nitro-o-phenylenediamine 2-nitro-p-phenylenediamine 1 ,3 ,S-triaminobenzene 2-hydroxy-pphenylenediamine 2,4-diaminobenzoic acid sodium 2,4-diaminobenzoate calcium di-2,4-diaminobenzoate ammonium 2,4-diaminobenzoate trimethylammonium 2,4-diaminobenzoate tri-(Z-hydroxyethyl) ammonium diaminobenzoate 2,4-diaminobenzaldehyde 2,4diaminobenzenesulfonic acid potassium 2,4-diaminobenzenesulfonate N,N-diisopropyl p-phenylenediamine N,N-dimethyl-p-phenylenediamine N-methyl-N'-(2-propenyl)-p-phenylenediamine N-phenyl-p-phenylenediamine N-phenyLN-benzyl-p-phenylenediamine Nethyl-N-(3-ethylphenyl)-p-phenylenediamine 2,4-toluenediamine Z-ethyl-p-phenylenediamine 2-(2-bromoethyl)-p-phenylenediamine 2phenylp-phenylenediamine 4-(2,5-diaminophenyl)benzaldehyde 2benzyl-p-phenylenediamine 2-(4-nitrobenzyl)-p-phenylenediamine 2-(4-methylpheny1)-p-phenylenediamine 2-(2,5-diaminophenyl)-5-methylbenzoic acid where X is the same as in formula (A), R and R can be the same or different from each other and are the same as in formula (A), R is the same as in formula (A) and R is hydrogen or C to C alkyl or alkenyl (including substituted alkyl or alkenyl wherein the substituents are selected from those defined as X in formula (A), above). Specific examples of formula (B) compounds are:

7 N-methyl-N-(3-ethylphenyl)-p-aminophenol 2-nitro-S-ethyl-p-aminophenol 2-nitro-5-( 2-bromoethyl )-p-aminophenol (2-hydroxy-5-aminophenyl)acetaldehyde Z-methyl-p-aminophenol v (2-hydroxy-5-aminophenyl)acetic acid 3-(2-hydroxy-5-aminophenyl)-l-propene 3-( 2-hydroxy-5-aminophenyl )-2-chlorol-propene 2-phenyl-p-aminophenol 2-(4-nitrophenyl)-p-aminophenol 2-benzyl-p-aminophenol 2-(4-chlorobenzyl)-p-aminophenol 2-(4-methylphenyl)-p-aminophenol 2-(2-amino-4-methylphenyl)-p-aminophenol p-methoxyaniline 2-bromoethyl-4-aminophenol ether 2-nitroethyl-4-aminophenyl ether 2-aminoethyl-4-aminophenyl ether 2hydroxyethyl-4-aminophenyl ether (4-aminophenoxy)acetaldehyde (4-aminophenoxy)acetic acid (4-aminophenoxy)methanesulfonic acid l-propenyl-4-aminophenyl ether (2-chloro l propenyl-4-aminophenyl ether (2-nitro 1 propenyl-4-aminophenyl ether (2-amino)- l-propenyl-4-aminophenyl ether (2-hydroxy)- l propenyl-4-aminophenyl ether where X, R and R are defined above in formula (B). Specific examples of formula (C) compounds are:

o-hydroxyphenol m-hydroxyphenol p-hydroxyphenol 4-methoxyphenol 2-methoxyphenol 4-(2-chloroethoxy)phenol 4-(2-propenoxy)phenol 4-(3-chloro-2-propenoxy)phenol 2-chloro-4-hydroxyphenol 2-nitro-4-hydroxyphenol 2-amino-4-hydroxyphenol 1,3 ,5-trihydroxybenzene 2,4-dih'ydroxybenzaldehyde 3,4-dihydroxybenzaldehyde 3,4-dihydroxybenzoic acid 2,4-dihydroxybenzenesulfonic acid 3-ethyl-4-hydroxyphenol 3-(2-nitroethyl)-4-hydroxyphenol 3-( 2-propenyl 1 hydroxyphenol 3-( 3-chloro-2-propenyl )-4-hydroxyphenol 2-phenyl-4-hydroxyphenol 2-(4-chlorophenyl)-4-hydroxyphenol 2-benzyl-4-hydroxyphenol 2-( 2-nitrophenyl )-4-hydroxyphenol 2-( 2-methylphenyl )-4-hydroxyphenol 2-( 2-methyl-4-chlorophenyl)-4-hydroxyphenol 3methoxy-4-hydroxy-benzaldehyde 2-methoxy-4-( l-propenyl)phenol 4-hydroxy-3methoxycinnamic acid 2,5-dimethoxyaniline The secondary oxidation dye precursors are optionally employed in the process of this invention and include those aromatic amines and phenols and derivatives thereof which do not produce color formation in the above test but which modify the color, shade or intensity of color developed by primary dye precursors. Various aromatic amines and phenolic compounds, and derivatives thereof, including aromatic diamines and polyhydric phenols of the types described by formulas (A), (B) and (C) above, but which are found by the above test not to be suitable primary oxidation dye precursors, are suitable as secondary dye precursors if they are capable of modifying the color, shade or intensity of color produced by primary oxidation dye precursors in the following test, which is conducted at room temperature (about 18 to 28C.).

SECONDARY DYE PRECURSOR TEST Two solutions are prepared as follows:

10 ml of aqueous buffer (pH 5 to 8) containing 0.01% to 1.0% (by weight) H 0 is mixed with 0.l to 1.0 ml of a 1% (by weight) acqueous of alcoholic solution of primary precursor. 0.1 to 1.0 ml. of a 1.0% (by weight) aqueous or alcoholic solution of the potential secondary dye precursor is added to one of the solutions and then an amount of horseradish peroxidase is added to each solution such that the final enzyme concentration is 0.01 to ppm (based on weight of pure enzyme). The solutions are left to stand for 5 minutes to permit development of color. A suitable secondary oxidation dye precursor will cause the second solution to differ from the first solution in color, shade or intensity of color.

The aromatic amines and phenols and derivatives described above as secondary oxidation dye precursors can also have additional substituents on the aromatic ring, e. g., halogen, aldehyde, carboxylic acid, nitro, sulfonyl and substituted and unsubstituted hydrocarbon groups, as well as additional substituents on the amino nitrogen, or phenolic oxygen, e.g., substituted and unsubstituted alkyl and aryl groups.

Examples of aromatic amines, phenols and derivatives thereof are compounds of the general formulas (D) and (E) below:

Rl N R 2 wherein Z is hydrogen, C to C alkyl, halogen (e.g., fluorine, chlorine, bromine or iodine) nitro,

COOM or SO M, (where M is hydrogen or an alkali or alkaline earth metal, ammonium or substituted ammonium wherein one or more hydrogens on the ammonium ion is replaced with a l to 3 carbon atom alkyl or hydroxy alkyl radical). wherein R, and R are the same or different and are each selected from the group consisting of hydrogen, C to C alkyl or alkenyl and C to C aryl, alkaryl or aralkyl and R is hydrogen, C to C alkyl or alkenyl (including substituted alkyl or alkenyl wherein the substituents are selected'from those designated as Z. above) or C to C aryl, alkaryl or aralkyl (including aryl, alkaryl or aralkyl having substituents selected from those defined as Z, above). Specific examples of formula (D) compounds are:

aniline p-chloroaniline p-fluoroaniline p-nitroaniline p-aminobenzaldehyde p-aminobenzoic acid sodium-p-aminobenzoate lithium-p-aminobenzoate calcium di-p-aminobenzoate ammonium-p-aminobenzoate v trimethylammonium-p-aminobenzoate tri( Z-hydroxyethyl )-p-aminobenzoate p-aminobenzenesulfonic acid potassium p-aminobenzenesulfonate N methylaniline N-propyl-N-phenylaniline N-methyl-N-2-propenylaniline N-benzylaniline' N-(2-ethylphenyl)aniline 4-methylaniline 4-( 2-bromoethyl)aniline 2-(2-nitroethyl)aniline (4-aminophenyl)acetaldehyde (4-aminophenyl)acetic acid 4-(2-propenyl)aniline 4-( 3-bromo-2-propenyl )aniline 4-phenylanilin'e 4-( 3-chlorophenyl )aniline 4-benzylaniline 4-(4-iodobenzyl)aniline 4-(3-ethylphenyl)aniline 4-( 2-chloro-4-ethylphenyl )aniline (a) I R7 Z wherein Z and R are defined as in formula (D) and R is hydrogen or C, to C alkyl or alkenyl (including substituted alkyl or alkenyl wherein the substituents are selected from those defined as Z in formula (D), above). Specific examples of formula (E) compounds are:

phenol p-chlorophenol p-nitrophenol phydroxybenzaldehyde p-hydroxybenzoic acid p-hydroxybenzenesulfonic acid ethylphenyl ether 2-chloroethylphenyl ether 2-nitroethylphenyl ether phenoxyacetaldehyde phenoxyacetic acid 3-phenoxy- 1 -propene 3-phenoxy-2-nitrol -propene 3-phenoxy-2-bromol -propene 4-propylphenol 4-(3-bromopropyl)phenol 2-( 2-nitroethyl )phenol (4-hydroxyphenyl)acetaldehyde (4-hydroxyphenyl)acetic acid 4-(2-propenyl)phenol 4-phenylphenol 4-(3-bromophenyl)phenol 4-benzylphenol 4-(3-fluoro-2-propenyl)phenol 4-(4-chlorobenzyl)phenol 4-( 3 -ethylphenyl )phenol 4-(2-chloro-3-ethylphenyl)phenol 2,5-xylenol I Primary oxidation dye precursors can be used alone or in combination with other primary oxidation dye precursors, and one or more primary oxidation dye precursors can be used in combination with one or more secondary oxidation dye precursors. The choice of a single oxidation dye precursor or of a particular combination of oxidation dye precursors will be determined by the color, shade and intensity of coloration which is desired. The total concentration of oxidation dye precursor in the coloring solution can be from about 0.001% to about 6% by weight and is preferably from about 0.01% to about 2.0% by weight.

The heme complexing agents contemplated by the present invention are characterized by an unshared pair of electrons on a nitrogen atom. It is believed that the nitrogen atom forms a complex with the ferric iron of the heme group of the peroxidase enzyme resulting in protection of the ferric iron from the reducing action of the anti-oxidant. Peroxidase enzyme is inactivated,

by the reduction of the ferric iron.

Preferred heme complexing agents are imidazole and benzimidazole'but many other heme complexing agents are effective to stabilize peroxidase enzymes in accordance with the present invention. Examples of other effective heme complexing agents are pyridine and its derivatives, ammonia, where the substituent is an alkyl group of from 1 to 3 carbon atoms, substituted imidazoles where the substituent is an alkyl group of from l,to 3 carbon atoms, benzotriazole, substituted benzotriazoles, where the substituent is an alkyl group of from 1 to 3 carbon atoms, 1,2,3-triazole, 1,2,4-triazole, pyrazole, Z-isoimidazole, pyrrole, and compounds of the general formula wherein R,, R and R are the same or different from each other, are selected from the group consisting essentially of hydrogen, alkyl groups of from 1 to 3 carbon atoms, hydroxyalkyl groups of from 1 to 3 carbon atoms, and any two of R R and R may be connected to make a cyclic compound.

The anti-oxidant agents contemplated by the present invention are those anti-oxidants which are more easily substituted benzimidazoles oxidized than the dye precursor but which will not displace the nitrogenous ligand which has formed a complex with the enzyme. Examples of anti-oxidants of the present invention are sodium sulfite, sodium dithionite, thioglycolate, and ascorbic acid. Preferred antioxidants are sodium sulfite and sodium dithionite. A solution of this invention comprises from about 0.1% to about 1% by weight of an anti-oxidant compound.

Hair coloring products employing oxidation hair dyes are typically marketed in kit form, i.e., a package comprising an individually packaged oxidizing component and an individually packaged oxidation dyeing component. In an embodiment of this invention the oxidizing component comprises an aqueous solution of from about 0.1% to about 6% by weight of hydrogen peroxide, and the oxidation dyeing premix component comprises from about 0.001% to about 6% by weight of one or more primary oxidation dye precursors or one or more secondary oxidation dye precursors or a mixture thereof, from about 0.005 ppm to about 500 ppm of a heme peroxidase enzyme, from about 0.1% to about 1% by weight of an anti-oxidant selected from the group consisting essentially of sodium sulfite, sodium dithionite, thioglycolate and ascorbic acid, and from about 0.01% to about 1% by weight of a nitrogenous ligand. The oxidizing component and the oxidation dyeing premix component are mixed by the user on the hair whereupon the hair is colored.

EXAMPLE I This example illustrates the coloring of human hair with a composition of the present invention. All percentages are by weight. 60 grams of a dilute hydrogen peroxide solution buffered to pH 6 are prepared by dissolving 2 grams of an aqueous 30% hydrogen peroxide solution in 58 grams of an aqueous NaH PO /Na HPO buffer solution, which is 0.1 M in phosphate. 60 grams of a dye base premix composition of the invention are prepared consisting of 1% p-phenylenediamine, 0.005% horseradish peroxidase, 0.25% sodium sulfite, 0.1% benzimidazole, 5% ethanol (solvent and wetting agent), 0.2% Dow Corning EF-l3574A (a cationic polysiloxane conditioner from the Dow Corning Company), Varion CDG (a coco betaine surfactant from the Northern Petrochemical Company), 1% JR-IL (a cationic cellulose derivative thickener from Union Carbide Company having the structure wherein R is a residue of an anhydrous glucose unit wherein y is an integer of 50 to 20,000 and wherein each R individually represents a substituent of the general formula wherein m is an integer ofO to 10, n is an integer from 0 to 3, and p is an integer from 0 to 10. The average values per anhydroglucose unit are: n is from 0.35 to 0.45 and the sum of m p is from 1 to 2) and the balance water containing a pH 6 buffer as described above. The premix composition is combined with hydrogen peroxide on the hair of a human female subject having naturally light brown hair by working it into a rich lather which remains on the hair and does not run down the neck and forehead. After working the mixture for 1 minute (to insure uniform application to all of the hair), the foamy lather is allowed to remain on the hair an additional 3 minutes. The subject then rinses her hair thoroughly with tap water and allows it to dry. It is observed that the hair has changed from its original light brown color to a medium brown shade.

In the above example, the horseradish peroxidase is replaced by an equivalent amount of NAD peroxidase, NADP peroxidase, fatty acid peroxidase, soybean hull peroxidase, cytochrome peroxidase, iodinase, and glutathione peroxidase, respectively, and equivalent results are secured.

In the above example, the p-phenylenediamine is replaced by an equivalent amount of ophenylenediamine, 2,4-toluenediamine, N-phenyl-pphenylenediamine, N,N-dimethyLp-phenylenediamine, 4-nitro-o-phenylenediamine, o-hydroxyphenol, phydroxyphenol, 3,4-dihydroxybenzaldehyde, 2- methoxy-4-(l-propenyl)phenol, 4-methoxyphenol, pmethoxyaniline, N,N-dimethyl-p-phenylenediamine, 4-hydroxy-3-methoxycinnamic acid, o-aminophenol, m-aminophenol, p-aminophenol, 2-nitri-paminophenol, 2-amino-l-phenol-4-sulfonic acid, and mixtures thereof, respectively, and shampoo-fast hair colors are secured.

In the above example, benzimidazole is replaced by an equivalent amount of imidazole, pyridine, ammonia, benzotriazole, 1,2,3-triazole, 1,2,4-triazole, pyrazole, 2-isoimidazole, and pyrrole and equivalent results are obtained.

EXAMPLE II 60 grams of a dilute hydrogen peroxide solution buffered to pH 6 are prepared as in Example I. 60 grams of a dye base premix composition of the invention are prepared consisting of 1% nitro-p-phenylenediamine, 1% p-aminophenol, 0.015% soybean hull peroxidase, 0.1% imidazole, 10% Miranol S2M (a dicarboxylic capric imidazoline which is an amphoteric surfactant from the Miranol Chemical Company having the structure) 0.5% sodium sulfate, and 2% Klucel G (a hydroxypropyl cellulose thickener from Hercules, Inc. having the structure) OH |)c|-| c|-|c|-| 3 l CH2 H OCH2CHCH3 H 1 l l H o d H OH OCH CHCl-l H o o I H H H 1 OC H' Ci-ICH CH2 oH OCH2|CHCH3 ocl-l izncn in a pH 6 buffer as described inExample I. The premix is combined with hydrogen peroxide on the hair of a human female subject having naturally light brown hair as in Example I. The subject then rinses her hair thoroughly with tap water and allows it to dry whereupon it is observed that the hair has changed from .its original color to an auburn color.

EXAMPLE III Procter & Gamble Company), and 0.75 g. of Na H- PO /NaH PO (pH 6 buffer). The oxidation dyeing component is admixed with the hydrogen peroxide and the solution is applied to the hair and provides a brownblack color which is substantially shampoo-stable.

What is claimed is:

1. A premix solution for dyeing hair comprising:

A. from about 0.005 ppm to about 500 ppm ofa peroxidase enzyme;

B. from about 0.01 to about 1% of a nitrogenous ligand which is a heme complexing agent selected from the group consisting of imidazole, benzimidazole, pyridine, ammonia, benzotriazole, 1,2,3- triazole. 1.2,4-triazole, pyrazole, 2-isoimidazole and pyrrole;

C. from about 0.1 to about 1% of an anti-oxidant selected from the group consisting of sodium sulfite, sodium dithionite, thioglycolate and ascorbic acid;

D. from about 0.01 to about 6% of an oxidation dye precursor selected from the group consisting of a primary oxidation dye precursor, a secondary dye precursor and mixtures thereof; and

E. balance. water, said composition having a pH of from about 4 to about 10.

2. A premix solution for dyeing hair according to claim 1, wherein the peroxidase enzyme is selected from the group consisting of NAD peroxidase, fatty acid peroxidase. NADP peroxidase, cytochrome peroxidase. horseradish peroxidase, iodinase, glutathione peroxidase. and soybean hull peroxidase.

3. A premix for dyeing hair according to claim 1 wherein the anti-oxidant is selected from the group consisting of sodium sulfite and sodium dithionite.

4. A premix solution for dyeing hair according to claim "1' wherein said primary oxidation dye precursor is selected from the group consisting of:

N R R and R5 N R R wherein X is selected from the group consisting of hydrogen, halogen, nitro, amino, hydroxyl,

o 0 II H CH COM and SO M where M is hydrogen, alkali or alkaline earth metal, ammonium or substituted ammonium wherein one or more hydrogens on the ammonium ion and wherein R R and R are each selected from the group consisting of hydrogen, C, to C alkyl and alkenyl and aryl, alkaryl, and aralkyl having 6 to 9 carbon atoms, R. is selected from the group consisting of hydrogen, alkyl and alkenyl groups having from 1 to 4 16 carbon atoms, Z is selected from the group consisting of hydrogen, C to C alkyl, halogen, nitro,

ll -CH COOM and -SO M where M is hydrogen or an alkali or alkaline earth metal, ammonium or substituted ammonium wherein one or more hydrogens on the ammonium ion is replaced with a l to 3 carbon atom alkyl or hydroxyalkyl radical.

6. A hair dyeing composition comprising:

A. from about 0.005 ppm to about 500 ppm ofa peroxidase enzyme;

B. from about 0.01 to about 1% of a nitrogenous ligand which is a heme complexing agent selected from the group consisting of imidazole, benzimidazole, pyridine, ammonia, benzotriazole, 1,2,3- triazole, 1,2,4-triazole, pyrazole, 2-isoimidazole and pyrrole;

C. from about 0.1 to about 1% of an anti-oxidant selected from the group consisting of sodium sulfite, sodium dithionite, thioglycolate, and ascorbic acid;

D. from about 0.01 to about 6% of an oxidation dye precursor selected from the group consisting of a primary oxidation dye precursor, a secondary dye precursor and mixtures thereof;

E. from about 0.01 to 6% of hydrogen peroxide; and

F. balance water, said composition having a pH of from about 4 to about 10. 

1. A PREMIX SOLUTION FOR DYEING HAIR COMPRISING: A. FROM ABOUT 0.005 PPM TO ABOUT 500 PPM OF A PEROXIDASE ENZYME, B. FROM ABOUT 0.01 TO ABOUT 1% OF A NITROGENOUS LIGAND WHICH IS A HEME COMPLEXING AGENT SELECTED FROM THE GROUP CONSISTING OF IMIDAZOLE, BENZIMIDAZOLE, PYRIDINE AMMONIA, BENZOTRIAZOLE, 1,2,3-TRIAZOLE, 1,2,4-TRIAZOLE, PYRAZOLE, 2-ISOIMIDAZOLE AND PYRROLE, C. FROM ABOUT 0.1 TO ABOUT 1% OF AN ANTI-OXIDANT SELECTED FROM THE GROUP CONSISTING OF SODIUM SULFITE, SODIUM DITHIONITE, THIOGLYCOLATE AND ASCORBIC ACID, D. FROM ABOUT 0.01 TO ABOUT 6% OF AN OXIDATION DYE PRECURSOR SELECTED FROM THE GROUP CONSISTING OF A PRIMARY OXIDATION DYE PRECURSOR, A SECONDARY DYE PRECUSOR AND MIXTURES THEREOF, AND E. BALANCE WATER, SAID COMPOSITION HAVING A PH OF FROM ABOUT 4 TO ABOUT
 10. 2. A premix solution for dyeing hair according to claim 1, wherein the peroxidase enzyme is selected from the group consisting of NAD peroxidase, fatty acid peroxidase, NADP peroxidase, cytochrome peroxidase, horseradish peroxidase, iodinase, glutathione peroxidase, and soybean hull peroxidase.
 3. A premix for dyeing hair according to claim 1 wherein the anti-oxidant is selected from the group consisting of sodium sulfite and sodium dithionite.
 4. A premix solution for dyeing hair according to claim 1 wherein said primary oxidation dye precursor is selected from the group consisting of:
 5. A premix solution for dyeing hair according to claim 1 wherein said secondary dye precursor is selected from the group consisting of:
 6. A hair dyeing composition comprising: A. from about 0.005 ppm to about 500 ppm of a peroxidase enzyme; B. from about 0.01 to about 1% of a nitrogenous ligand which is a heme complexing agent selected from the group consisting of imidazole, benzimidazole, pyridine, ammonia, benzotriazole, 1,2,3-triazole, 1,2,4-triazole, pyrazole, 2-isoimidazole and pyrrole; C. from about 0.1 to about 1% of an anti-oxidant selected from the group consisting of sodium sulfite, sodium dithionite, thioglycolate, and ascorbic acid; D. from about 0.01 to about 6% of an oxidation dye precursor selected from the group consisting of a primary oxidation dye precursor, a secondary dye precursor and mixtures thereof; E. from about 0.01 to 6% of hydrogen peroxide; and F. balance water, said composition having a pH of from about 4 to about
 10. 